Glaucoma, the leading cause of irreversible blindness in the world; currently affects more than 3 million in the U.S. and is projected to increase to ~6.3 million by 2050. The four major types of adult-onset glaucoma all lead to vision loss through a final common pathway of retinal ganglion cell (RGC) dysfunction and/or death. The current standard of care includes treatment with IOP-lowering eye drops, all of which have limited efficacy. Specifically, IOP reduction does not fully prevent RGC death and resulting visual field loss in many glaucoma patients. In our previous NIH-funded studies, we identified both a novel IOP-lowering drug target, the calcium channel, voltage-dependent, α2δ1subunit (aka CACNA2D1) and a selective CACNA2D1 blocker, pregabalin (PRG), that lowers IOP. Lastly, we developed a topical extended release PRG microemulsion (ME) that increases drug entry into the eye, resulting in higher efficacy and duration of action. Our most recent studies have uncovered an unanticipated additional treatment benefit of PRG, namely its additional localization to, and neuroprotection of, RGCs and optic nerve (ON) as we have demonstrated in an animal model of normal tension glaucoma. Thus, PRG ME could be the first glaucoma therapy to both lower IOP and have direct, neuroprotective effects on RGCs and their axons. This current SBIR Phase I application is designed to validate our new findings regarding the neuroprotective effects of our PRG ME, and to establish its mechanism of action. Our central hypothesis is that our PRG ME will lower IOP with improved efficacy, as well as maintain the health of RGCs, a field of use for which there is currently no FDA-approved drug. This hypothesis is supported by our data showing that CACNA2D1 is located in RGCs and ON and that topical administration of our extended-release bioadhesive ME promotes delivery of PRG to the retina. Overall strengths of this project include: 1) a strong and experienced interdisciplinary OculoTherapy team; 2) engineering of an innovative delivery strategy using an extended-release formulation; 3) the use of a highly promising FDA-approved drug that will be repurposed as a glaucoma therapeutic; and 4) the discovery that the drug may have multiple sites of action within the eye that collectively provide IOP lowering through direct interactions with the ciliary body and/or outflow structures as well as direct RGC neuroprotection that is independent of its IOP-lowering capability. In this Phase I SBIR proposal, we will provide proof of concept and address key feasibility questions by establishing neuroprotective efficacy and mechanism of action of our PRG ME. This treatment strategy will reduce the burden to the patient and lead to better visual outcomes for glaucoma patients. To address our objective, we propose the following Aims: Aim 1: We test the hypothesis that PRG is a neuroprotectant for RGCs and the ON using once daily dosing. Aim 2: We test the hypothesis that PRG plays a dir...